Use of (3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole for the treatment of autoimmune diseases

ABSTRACT

The present invention discloses a method for treating a subject affected by an autoimmune disease, in particular multiple sclerosis, lupus erythematosus systemicus and rheumatoid arthritis, comprising administering to said subject an effective amount of 3-(2-ethylphenyl)-5-methoxy-1H-1,2,4-tirazole. The present invention further discloses a method for inhibiting γδ T cells in a subject in need thereof, said method comprising administering to said subject an effective amount of the same compound.

The present invention relates to a method for the treatment ofautoimmune diseases, which are effectively treated by administering thecompound (3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is an inflammatory demyelinating disease of thecentral nervous system (CNS) that is thought to be mediated by anautoimmune attack directed against CNS myelin antigens. Based on animalmodels, as well as on data gathered from analyses of leukocytes andtissues from patients with MS, antigen specificity is considered toreside within T cells expressing the αβ T cell receptor (TCR), withencephalitogenic activity dependent on the expression of a cytokineprofile characteristic of a Th1-type phenotype: interferon-gamma (IFNγ),lymphotoxin (LT) and tumor necrosis factor-alpha (TNFα). T cells thatexpress a Th2-type cytokine profile (IL-4, IL-5 and IL-13) or regulatorycytokines such as transforming growth factor-beta (TGFβ) and IL-10, arethought to interfere with this process by blocking the acquisition ofthe Th1-phenotype and/or by blocking the downstream targets of effectorcytokines, such as the activation of macrophages.

The critical nature of the cytokine profile of the antigen-reactive αβ Tcells to disease expression raises the question as to the nature of theantigen recognition process that results in the acquisition of thesespecific cytokine profiles. Although it has been known for some timethat the presence of adjuvants and the route of antigen presentation areimportant determinants of encephalitogenic activity in animals, howthese processes shape the nature of the acquired immune response, aswell as the contribution of other non-antigen-specific leukocytepopulations, have only recently started to come into sharper focus withan expanding recognition of the different cell populations that functionat the interface between the innate and acquired immune response.

The innate immune response functions as a first line of defense againsta wide range of infectious and toxic agents. Historically, this responsehas been attributed to cells with phagocytic activity, such asmacrophages and polymorphonuclear cells, and/or potent cytotoxicactivity, such as natural killer cells (NK cells), mast cells andeosinophils. The activity of these different cell populations is aidedand abetted by a number of different soluble molecules collectivelyknown as acute phase proteins, such as the interferons, specificcomponents of the complement cascade and cytokines, that serve toenhance phagocytic and cytotoxic activity, as well as lead to theaccumulation of these cells at sites of tissue injury. If these firstlines of defense are breached, then activation of the adaptive immuneresponse ensues, leading to the formation of a specific immune responsethat may display anyone of a number of different characteristics. Thegeneration of this acquired immune response is an exclusive property oflymphocytes.

More recently, however, it has become recognized that minorsubpopulations of lymphocytes may also function as part of the innateimmune response. Although it is likely that the complete functional roleof these specialized subsets of lymphocytes remains poorly understood,current interest in them has focused on their role in defining thecytokine milieu at sites of tissue injury, influencing the nature of theadaptive immune response that is generated. Thus, many of these studieshave focused on the role of IFNγ and IL-12 in defining a Th1-typecytokine profile and IL-4 a Th2-type cytokine profile. Subpopulations ofall of the three major groups of lymphocytes, αβ T cells, γδ T cells andB cells, likely fall into this category. These lymphocyte populationsare characterized by the use of a highly conserved antigen receptorcomplexes, expression of additional pattern-recognition receptors, suchas members of the Toll-like receptor family or receptors normallydetected on NK cells, and the rapid release of high levels of cytokinesand chemokines following interaction with specific ligands.

γδ T cells: T cells expressing the γδ T cell receptor (TCR) constitute aminor population of the total circulating T cell population. In commonwith αβ T cells, γδ T cells express a rearranged TCR, but the mechanismsinvolved in the acquisition of TCR diversity, as well as the nature ofthe antigens recognized, are clearly different (Chien Y. H. et al. Annu.Rev. Immunol. 1996; 14:511-32). Analysis of CDR3 length distributions,as well as crystallographic studies, have suggested greater structuralsimilarity of the γδ TCR to immunoglobulin heavy chain genes, lendingfurther support to the conclusion that the molecular nature of antigenrecognition by γδ T cells is fundamentally different from that utilizedby αβ T cells. γδ T cells also differ from αβ T cells in that most γδ Tcells coexpress receptors found on natural killer cells (NK-R)(Battistini L. et al.; J. Immunol. 1997; 159:3723-30). Expression ofthese receptors on T cells has been shown to modulate several T cellfunctions including cytotoxicity, cytokine release and transendothelialcell migration (Reyburn H. et al., Immunol. Rev. 1997; 155:119-25).These data indicate that the regulation of γδ T cell function is likelyto be different from that found in most αβ T cells, involving activation(or inhibition) by signaling through both the TCR and NK-R. It has beensuggested that NK-R functions as costimulatory molecules that areexquisitely responsive to changes in cell surface expression of MHCmodulated by infection or to the activation state of the cells (ReyburnH. et al., ibid.).

In healthy adults the majority of γδ T cells express a TCR that utilizesthe Vγ9Vδ2 gene segments. The expansion of this specific population ofγδ T cells is thought to be due to a response to non-protein bacterialantigens such as pyrenil-pyrophosphate derivatives and other componentsof bacterial cell walls, without classical MHC-restriction (Salerno A.et al., Crit. Rev. Immunol. 1998; 18:327-57)). The response to thesetypes of antigens has been found to be critically dependent upon the useof germline encoded lysine residues in the Jγ1.2 segment (Miyagawa F. etal., J. Immunol. 2001; 167:6773-6779). Thus, although the response tophosphate antigens may be polyclonal in nature, conserved elements areused by the responding cells. Conserved sequences of γδ T cell receptorhave been noted in cells and/or tissues isolated from patients with MS,suggesting a response to a common antigen.

γδ T cells share many features in common with the αβTCR+NK-T cells,including the expression of NK receptors, constitutive expression of theIL-2rβ, usage of highly conserved TCR sequences and restriction, atleast for some subsets, by CD1 molecules (Spada F. M. et al., J. Exp.ed. 2000; 191:937-48). This would suggest that some γδ T cells mayprovide a similar link between the innate and acquired immune response(Poccia F. et al., Immunol. Today 1998; 19:253-6). Consistent with sucha notion is that activation of Vδ2+ T cells with phosphate antigens hasbeen shown to lead to the rapid release of large amounts of bothcytokines and chemokines (Poccia F. et al., J. Immunol. 1997;159:6009-17; Cipriani B. et al., Blood 2000; 95:39-47). Interestingly,there are accumulating data that suggest that V region usage mayimplicate specific subsets of γδ T cells in mediating Th1 or Th2-typeresponses—with Vδ2+ cells showing a Th1-type bias and Vδ1+ cells aTh2-type bias. So for example, it has been shown that γδ T cells in MSlesions express a predominantly Vδ2 phenotype (Battistini L. et al.,Mol. Med. 1995; 1:554-62) and that Vδ2 cells in the peripheral blood ofpatients with MS show evidence of activation. In the CSF, however, Vδ1cells are the predominant γδ T cell population (De Libero G., SpringerSemin. Immunopathol. 2000; 22:219-38).

Studies that have examined a potential role for γδ T cells indemyelinating diseases further support the conclusion that although γδ Tcells show evidence of activation in patients with either MS or GuillainBarrè syndrome (GBS), differences exist in the phenotypic and functionalproperties of these cells in the two diseases. In particular, the dataindicate that in patients with MS the Vδ2 subset is activated and thatthese cells can be induced to secrete high levels of proinflammatorycytokines.

Once activated via the TCR, 1γδ T cells may also function as NK cells,responding in either a positive or negative fashion to NK cell targets(Battistini L. et al., J. Immunol. 1997; 159:3723-30; De Libero G.Microbes Infect. 1999; 1:263-7). Furthermore, in MS patients with activedisease, the percentage of circulating Vδ2+ T cells coexpressing NKRP1A(the human homologue of NK1.1) has been found to be significantlyincreased compared with healthy donors. When Vδ2+ and Vδ1+ T cells weresorted from MS patients and healthy volunteers and cloned, all Vδ2+clones expressed NKRP1A. NKRP1A was strongly up-regulated on Vδ2+ cellsby culture with IL-12 whereas no up-regulation of NKRP1A by IL-12 wasnoted on Vδ1+ clones. In transendothelial migration assays, Vδ2+ NKRP1A+clones migrated more effectively than Vδ1+ clones, and this migratorypotential was enhanced following culture with IL-12. Migration wasstrongly inhibited by the F(ab′)2 of an anti-NKRP1A antibody, suggestingthat this receptor for common lectins is involved in thetransendothelial cell migration process. It was also shown that infreshly isolated PBMC from MS patients, the migrated population wasenriched in Vδ2+ NKRP1A+ cells. Thus the expression of NKRP1A on Vδ2+cells is associated with an increased ability to migrate across thevascular endothelium, an activity that may be upregulated by IL-12present in the microenvironment (Poggi A. et al., J. Immunol. 1999;162:4349-54). Taken together, these data suggest that γδ T cells couldbe rapidly recruited to sites of inflammation in the CNS where theycould significantly contribute to the cytokine/chemokine balance of thelesion, as has been demonstrated in EAE (Spahn T. W. et al, Eur. J.Immunol. 1999; 29:4060-71; Rajan A. J. e al., J. Immunol. 2000;164:2120-30).

Accordingly the availability of a compound having immunomodulatoryproperties on the innate immune response of effector γδ+ T cells wouldbe of great benefit to the subjects in need thereof.

Abstract of the Invention

It has now been found that a compound of the 3,5-diaryl-s-triazolesclass of molecules, more precisely(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole (hereinafteralso called ST1959) efficiently treats autoimmune diseases. It has alsobeen found that the compound according to the present invention inhibitsthe γδ T cell effector response by a non-cytotoxic mechanism.

Accordingly, it is an object of the present invention a method fortreating a subject affected by an autoimmune disease comprisingadministering to said subject an effective amount of(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole

In particular, according to the method of the present invention, saidsubject is affected by an autoimmune disease, such as multiplesclerosis, lupus erythematosus sistemicus, arthritis reumatoid (RA).

In another aspect of the present invention, it is a further object ofthe present invention a method for inhibiting γδ T cells in a subject inneed thereof, said method comprising administering to said subject aneffective amount of(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole.

In a preferred embodiment of the present invention, said subject is amammal, more preferably a human.

(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole was described inU.S. Pat. No. 4,379,155, issued on 5 Apr. 1983, as part of a largefamily of 1,2,4-triazoles with anti-fertility activity. This compoundwas thoroughly studied as anti-fertility agent (Galliani et al. Pharm.Dyn. 5, 55-61 (1982)). Later, U.S. Pat. No. 6,323,230, assigned toGeange Ltd., issued on 27 Nov. 2001, discloses a large family ofnitrogen heterocyclic aromatic derivatives useful for the topicaltreatment of the epithelial tissues diseases, in particular psoriasis,atopic dermatitis, ulcerative colitis and Crohn's disease and theadministration routes are epicutaneous, oral and rectal. According tothe disclosure of the above mentioned U.S. Pat. No. 6,323,230, the oralroute is to be understood as an oral formulation suitable for deliveringthe compound specifically at the site of action, namely at theepithelial mucosae of the low intestine. In fact, the oral route, whenconsidered for the original anti-fertility activity, isn't the rightchoice with respect to the parenteral injection (Galliani et al. Pharm.Dyn. 5, 55-61 (1982)), due to a rapid and extensive hepatic first-passeffect leading to the formation of inactive metabolites (Assandri A. etal. Drug Interactions, IV, 237-261 (1982); Assandri A. et al.Xenobiotica 14, 429-433 (1984)). The same reference teaches that thecompound according to the present invention does not retain hormonal oranti-hormonal or lympholytic activity, inhibits the antibody formationversus corpuscolar antigens (ram erythrocytes) when administered afterthe agents, does not exert a selective action on lymphocytes B and/or T.The immunological profile of the compound was described in Mistrello G.et al., Immunopharmacology, vol. 10, 1985, 163-169. In this reference,the compound of the present invention showed to be inactive in treatingarthritis.

The compound of the present invention has inhibitory activity on γδ Tcells, therefore, is useful in the treatment of diseases which areeffectively treated by administering a compound inhibiting γδ T cells.

The administration of the compound of the present invention is made bymeans of conventional pharmaceutical compositions, for example, asdisclosed in the above mentioned U.S. Pat. No. 4,379,155 and U.S. Pat.No. 6,323,230. The preferred route of administration, although notexclusive, is the subcutaneous one.

The following examples further illustrate the invention.

EXAMPLE 1 Multiple Sclerosis

Blood samples were obtained from 18 healthy volunteers and from 18patients with clinically active MS in the relapsing phase or in thefirst episode of disease, with abnormal magnetic resonance imaging brainscan; none had received immunosuppressive treatment for at least 3months before entering the study. Patients and 18 healthy donors werematched for sex and age

Peripheral and cord blood mononuclear cells were isolated fromheparinized blood by Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden)and cultured at 1.5×10⁶ cells/ml in complete medium (RPMI 1640, 10% v/vheat-inactivated FCS, 2 mM L-Glutamine, 10 U/mlpenicillin/streptomycin). PBMC from control donors or MS patients werestimulated in vitro for 9 days in the presence of 30 μM isopentenylpyrophosphate (IPP; Sigma-Aldrich, St. Louis, Mo.) and 50 U/ml rIL-2(Boehringer Mannheim, Mannheim, Germany). After 3 days of culture, thevolume corresponding to half-culture supernatant was replaced bycomplete medium with rIL-2. The expansion of Vγ9Vδ2+ T cells after 6days of culture was determined by cytometric analysis using doublestaining with anti-CD3 and anti-TCR-Vδ2 mAbs coupled to PE or FITC,respectively. Vδ2 expansion index was calculated dividing the absolutenumber of Vδ2+ T cells in stimulated cultures by the absolute number ofVδ2+ T cells in unstimulated cultures.

Cytokine production was detected by flow cytometric analysis aspreviously described. Human PBMC were stimulated for 6 h with IPP (100μM; Sigma-Aldrich) and/or 100 U/ml rIL-2 (Boehringer Mannheim).Brefeldin A (10 μg/ml) was added 1 h after stimulation to blockintracellular transport allowing cytokine accumulation in the Golgi.Cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide andstained with mAbs specific for the membrane Ags described above for 15min at 4° C. Samples were then fixed in 1% paraformaldehyde for 10 minat 4° C., incubated with anti-IFN-mAb diluted in 1×PBS, 1% BSA, and 0.5%saponin. The cells were finally washed twice in 1×PBS, 1% BSA, 0.1%saponin, and acquired on a FACScan (BD Biosciences). Control fornonspecific staining was monitored with isotype-matched mobs andnonspecific staining was always subtracted.

IFN-γ levels were determined by a standard sandwich ELISA as previouslydescribed. Antibodies and standards were purchased from PharMingen.Enhanced protein-binding ELISA plates (Nunc Maxisorb; Nunc Maxi Corp.,Roskilde, Denmark) were used.

A rather unique feature of the Vγ9Vδ2 TCR is its ability to recognizeboth naturally occurring and synthetic non-peptidic phosphoantigens.These antigens can be found in pathogenetic microorganisms such asPlasmodium, Francisella, and Mycobacterium. One of these is isopentenylpyrophosphate (IPP), a 246-Da molecule that has a five-carbon isoprenylchain and a pyrophosphate moiety. Following activation by thesecompounds, Vγ9Vδ2 cells expand and rapidly secrete proinflammatorycytokines such as TNF-α and IFN-γ and acquire potent cytotoxic activity,implicating these cells as important mediators of inflammation at sitesof Ag recognition. It is known that IPP exclusively stimulatesproliferation of the Vδ2Vγ9 T-cell subset and also induces cytokineproduction in the same γδ T-cell population. To determine whether(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole inhibits Vδ2 Tcell expansion following IPP stimulation, freshly isolated PBMCs fromhealthy donors or MS patients were cultured with either IL-2, IL-2+IPP,or IL-2+IPP+(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole (30μM or 60 μM) and Flow Cytometric analysis was performed at day 6 afterstimulation to determine the percentage of cells present in the culturethat expressed the Vδ2 gene product. The compound of the presentinvention efficiently inhibited, in a dose response manner, theexpansion of Vδ2+ T cells when cultured together with IPP (FIG. 1). Thepercentages of inhibition of the expansion were 39% and 38%,respectively, when the compound was used at the concentration of 30 μMin cells isolated from MS patients or from healthy individuals. Adifference was noted in the inhibition caused by(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole at higherconcentration (60 μM) in the two groups studied, 71% of inhibition wasfound in cells isolated from MS patients versus 88% in cells fromhealthy individuals.

To determine whether the compound of the present invention was toxic forVδ2 T cells, unfixed cells were stained with PI to assess cell membraneintegrity and were analyzed by Flow Cytometry. In cells cultured for 6days with IL-2 alone 5.96% cells were PI +, whereas cells challengedwith IPP+ IL-2 showed 9.66% of PI+cells. When ST1958 (30 μM or 60 μM)was added PI+cells were respectively 15% and 17.65% (FIG. 2). The littleincrease PI+ dead cells in the cultures where(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole was addedcompared with that with IPP alone demonstrate that this compound lead toa robust inhibition of Vδ2 T cell expansion following IPP stimulation(56% of inhibition at 30 μM and 91% inhibition at 60 μM) by anon-cytotoxic mechanism (FIG. 2).

To determine whether(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole inhibited therelease of proinflammatory mediators by Vδ2 T cells activated withphosphate Ags, PBMCs were activated with IPP (30 μM) in the presence orthe absence of the compound at 30 μM and 60 μM, and the release of IFN-γwas determined by ELISA 24 h post-stimulation. The presence of thecompound in the medium potently inhibited the release of IFN-γ fromthese cells in a dose-dependent manner (55% and 65% of inhibition whenthe compound at 30 μM was added in the cells isolated from MS andhealthy donors respectively; 74% and 82% of inhibition when the compoundat 60 μM was added in the cells isolated from MS and healthy donorsrespectively—FIG. 3).

The effect of (3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazolewas tested on the induction of Vδ2+ IFN-γ+cells T cells followingstimulation for 6 h with IPP). These data show that the compound of theinvention determines a net reduction in a dose dependent manner of thenumber of Vδ2+ T cells producing IFN-γ following stimulation with IPP.

(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole used at aconcentration of 30 μM leads to inhibitions of 50% and 25% of the Vδ2+IFN-γ+cells induced by IPP in cells isolated from healthy donors and MSpatients, respectively. On the contrary the level of inhibition foundwith 60 μM of the compound was similar for the two group of patients:93% of inhibition of the Vδ2+ IFN-γ+cells induced by IPP in cellsisolated from healthy donors and 92% in cells isolated from MS patients(FIG. 4).

(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole has inhibitoryeffects on γδ T cells from both MS patients and healthy individuals. Inall situations tested there is a slight—but significant—difference inthe inhibitory effects exploited on cells derived from MS patientscompared to those derived from healthy individuals, in that inhibitionof γδ T cell function is more effective in healthy subjects. Poggi, A.et al., J. Immunol. 162:4349 have previously described that γδ T cellsin MS patients are more activated than those derived from healthyindividuals, as they express molecules which are critical forinteraction with endothelium and thus can readily migrate in inflamedtissue amplifying the inflammatory response which underliesdemyelination and, consequently, neurological disfunction. It isconceivable that inhibition of a pre-activated cell is more arduous thanthat of a resting population, since it requires the shutting off ofcellular mechanisms which have already been initiated. Nevertheless, inmost assays inhibition of γδ T cell function in MS patients reached60-70%. Given the wide distribution of the Ags recognized by theseVγ9Vδ2+ T cells and the rapidity with which proinflammatory cytokinessuch as IFN-γ and TNF-α and chemokines such as MIP-1α and MIP-1β areproduced through pathways that appear to differ from αβ T cells, thesecells could play an important role in the transition from the innate tothe acquired immune response by biasing reactions toward a Th1-typeresponse. This would suggest that at sites of Ag recognition(3-(2-ethylphenyl)-5-methoxyphenyl)-1H-[1,2,4]-triazole couldeffectively inhibit γδ T cell activation, which could have broadimplications for the activation of both innate and acquired immuneresponses.

EXAMPLE 2 Lupus

Mice MRL/lpr (female) of about 6 weeks were obtained from Jackson (USA).These mice spontaneously develop a Lupus like pathology around the 8thweek. ST1959 administration was started at the 6th week and performeds.c. twice/week 2.5 mg/kg in 0.1 ml of sesame oil. One group of treatedand one group of control (sesame oil) were included in the study (12mice/group). Once a week the renal damage was monitored by detectingproteinuria and urinary leucocytes. Data reported in the FIG. 5 arepresent the average of the % of animals with urinary proteins higherthan 100 mg/dl in the two groups. Panel b indicate the score of urinaryleucocytes (score 0-3). All determinations were performed by the use ofMultistix 10SG (UK). Panel c shows the % of survival of the two groupsand indicates a delay in start of mortality for the ST1959 treatedanimals compared to control group and a higher overall survival with 60and 25%, respectively. The admnistration of ST1959 did not affect thebody weight of treated animals as shown in panel d, thus suggesting lackof toxicity.

EXAMPLE 3 Collagen Induced Arthritis

Mice DBA/1J were obtained from Charles Rivers (Italy). Induction ofarthritis was performed by administration, at day 0 and +21 of 100μl/mouse i.d. of emulsions composed of equal volumes of CompleteFreund's adjuvant+2 mg/ml of M. tubercolosis and 4 mg/ml of bovine typeII collagen. Mice were randomly assigned to study groups (8 mice/group)including: ST1959 o.s. treated, cyclosporin (CSA) o.s. treated, vehicle(sesame oil). Sham mice were treated with emulsion lacking the collagenand did not receive further administrations. The compounds wereadministered 3 times/week s.c. or o.s. in 0.1 ml of sesame oil startingfrom day 0 or day +21. The scores of inflammation and anchilosis wasbetween 0-3 for each limb. As shown in FIG. 6, the ST1959 s.c. treatmentsignificantly reduced both inflammation (6a) and anchilosis (6b). On thecontrary, the oral administration did not produce any therapeuticeffect. The s.c. dose of ST1959 was {fraction (1/1500)} of its LD₅₀.Cyclosporine treatment, at a very high dose (100 mg/kg), was included asthe positive control.

EXAMPLE 4 Experimental Autoimmune Encephalomyelitis (EAE)

Female Lewis rats (7 weeks of age) were purchased from Harlan. Inductionof EAE was performed by injecting into each hind footpad 100 μg/200μl/rat of Myelin Basic Protein (MBP) emulsified in incomplete Freund'sadjuvant (200 μl/rat) containing heat killed Mycobacterium Tuberculosis(Mt) H34Ra, 350 μg/200 μl/rat (Hoban C. J., Exp. Opin. Ther. Patents, 8,7, 831-854, 1998; Ledeen R. W. et al., Neurochemical Research, 23, 3,277-289, 1998; Nagai H. et al., Gen. Pharmac., 30, 2, 161-166, 1998;Sommer N. et al., Nature Medicine, 1, 3, 1995; Simmonds S. et al.,Immunology Methods Manual, Academic Press Ltd, 1997).

Following the encephalitogenic challenge, mice were observed daily andclinical manifestations of EAE were scored on a scale ranging from 0 to6, as follows:

-   0: no clinical signs; 1: flaccid tail; 2: partial paralysis of hind    feet; 3: complete paralysis of hind feet; 4: paralysis of fore feet.

Rats were randomly assigned to study groups including: control(emulsion+MBP), sham (emulsion), treated (emulsion+MBP+ST1959subcutaneously in sesame oil 0.25, 0.50, 1.58 or 5.00 mg/5 ml/kg).ST1959 was administered daily from days 3 to 22 after immunization.

Control animals showed clinical signs of monophasic EAE from 10 daysafter immunization and complete recovery after 22 days; ST1959 reducedseverity, incidence and onset of disease (FIG. 7, 8) at four differentdoses.

EXAMPLE 5 Experimental Autoimmune Uveitis (EAU)

Male Lewis (rats 7 weeks of age) were purchased from Harlan. Inductionof EAU was performed by injecting into each hind footpad 125 μg/200μl/rat of synthetic human retinal antigen S—Ag emulsified in incompleteFreund's adjuvant (200 μl/rat) containing heat killed MycobacteriumTuberculosis (Mt) H34Ra (800 μg 200 μl/rat). Rats received BordetellaPertussis Toxin (1 μg/300 μl/rat in PBS) in the tail vein immediatelyafter immunization (Barton K. et al., Eye, 8, 60-65, 1994; Forrester J.V. et al., Chem. Immunol. Basel, Karger, 73, 159-185, 1999; Smith J. R.et al., Immunology and Cell Biology, 76, 497-512, 1998; Zamir E. et al.,Free Radical Biology & Medicine, 27 (1-2), 7-15, 1999).

Following the encephalitogenic challenge, mice were observed daily andclinical manifestations of EAU were scored as following:

-   No clinical signs: 0-   Iris hyperemia:    -   Low: 1    -   Mild: 2    -   Severe: 3-   Hypopyon:    -   Low: 1    -   Mild: 2    -   Severe: 3-   Maximal cumulative clinical score for both eyes: 12

Rats were randomly assigned to study groups including: control(emulsion+S—Ag), sham (emulsion), treated (emulsion+S—Ag+ST1959subcutaneously in sesame oil 0.50 mg/5 ml/kg). ST1959 was administereddaily from days 3 to 22 after immunization.

Control animals showed clinical signs of EAU from 10 days afterimmunization and amelioration but not complete recovery after 20 days;ST1959 reduced severity of disease at 0.50 mg/5 ml/kg (FIG. 9).

1-8. canceled.
 9. A method for treating uveitis in a subject in needthereof, comprising administering to said subject an effective amount of3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-triazole.
 10. The methodof claim 9, wherein said subject is a mammal.
 11. The method accordingto claim 9, wherein said subject is a human.